Detection of respiratory viruses

ABSTRACT

Specific nucleic acid sequences, e.g., SEQ ID NOs:1-57, for simultaneous detection of seven most common viruses that cause respiratory infections in human, i.e., human parainfluenza virus 1, human parainfluenza virus 2, human parainfluenza virus 3, respiratory syncytial virus, influenza virus A, influenza virus B, and adenovirus. Also disclosed is a method of simultaneously detecting these viruses. The method includes providing a nucleic acid prepared from a sample suspected of containing a virus to be detected, amplifying the nucleic acid with a set of primers specific for one or more of the seven viruses, and detecting amplification products. Detection of an amplification product specific for any one of the seven viruses indicates the presence of that particular virus.

BACKGROUND

Respiratory tract infections cause nearly half of the deaths due toinfectious diseases in the United States (Wei, et al., Obstet GynecolClin North Am (2001) 28 (2): 283-304). About 75 percent of acuterespiratory illnesses are caused by viruses. Human parainfluenza virus1, human parainfluenza virus 2, human parainfluenza virus 3, respiratorysyncytial virus, influenza virus A, influenza virus B, and adenovirusare the most common viruses that cause respiratory infections in bothchildren and adults. Detection of these viruses is essential fordiagnosis, prevention and treatment of respiratory diseases.

SUMMARY

The present invention relates to specific nucleic acid sequences forsimultaneously detecting multiple respiratory viruses including humanparainfluenza virus 1, human parainfluenza virus 2, human parainfluenzavirus 3, respiratory syncytial virus, influenza virus A, influenza virusB, and adenovirus.

In one aspect, this invention features a PCR primer set that containstwo primer pairs for detecting two respiratory viruses, humanparainfluenza virus 2 and adenovirus. One of the human parainfluenzavirus 2 primers contains an oligo-nucleotide selected from thehemagglutinin-neuraminidase gene region (e.g., SEQ ID NO:5 or 6), andthe other primer contains another oligo-nucleotide also selected fromthe same region (e.g., SEQ ID NO:7). One of the adenovirus primerscontains an oligo-nucleotide selected from the hexon gene region, andthe other primer contains another oligo-nucleotide also selected fromthe same region. For example, the oligo-nucleotides in an adenovirusprimer pair can be, SEQ ID NOs:24 and 26, SEQ ID NOs:24 and 27, or SEQID NOs:25 and 27. Each oligo-nucleotide has 14-40 (e.g., 14-35, 14-30,14-25, or 14-20) nucleotides in length.

The PCR primer set of this invention can further contain one or moreadditional specific primer pairs for detecting other respiratoryviruses. For instance, one or more of the following five primer pairs(including any combination thereof) can be included in the PCR primerset:

-   -   (1) a pair of human parainfluenza virus 1 primers, each        containing an oligo-nucleotide selected from the        hemagglutinin-neuraminidase gene region. For example, the        oligo-nucleotides in a human parainfluenza virus 1 primer pair        can be, respectively, SEQ ID NOS:1 and 3, SEQ ID NOs:2 and 3, or        SEQ ID NOs:1 and 4.    -   (2) a pair of human parainfluenza virus 3 primers, each        containing an oligo-nucleotide selected from the        hemagglutinin-neuraminidase gene region. For example, the        oligo-nucleotides in a human parainfluenza virus 3 primer pair        can be, respectively, SEQ ID NOs:8 and 10, SEQ ID NOs:8 and 11,        or SEQ IN NOs:9 and 11.    -   (3) a pair of respiratory syncytial virus primers, each        containing an oligo-nucleotide selected from the non-structural        protein 2 gene region. For example, the oligo-nucleotides in a        respiratory syncytial virus primer pair can be, respectively,        SEQ ID NOS:12 and 14, or SEQ ID 10 NOs:13 and 15.    -   (4) a pair of influenza virus A primers, each containing an        oligo-nucleotide selected from the non-structural protein gene        region. For example, the oligo-nucleotides in an influenza virus        A primer pair can be, respectively, SEQ ID NOS:16 and 18, or SEQ        ID NOS:17 and 19.    -   (5) a pair of influenza virus B primers selected from the        hemagglutinin gene region. For example, the oligo-nucleotides in        an influenza virus B primer pair can be, respectively, SEQ ID        NO:20 and 22, or SEQ ID NOs:21 and 23.

In another aspect, this invention features a set of nucleic acids thatcontains one or more nucleic acids obtained from amplification of agroup of respiratory virus nucleic acid templates with one of the PCRprimer sets described above. The group of nucleic acid templates isprepared from one or more of the seven viruses mentioned above. Theamplification products can be used as hybridization probes for virusdetection.

In yet another aspect, this invention features a probe set that can beused for detecting the seven respiratory viruses mentioned above. Eachprobe has 20-1,000 (e.g., 20-500, 20-200, or 20-50) nucleotides inlength. The probe set contains one or more of the following:

-   -   (1) a respiratory syncytial virus probe including an        oligo-nucleotide selected from the non-structural protein 2 gene        region. For example, the oligo-nucleotide can be one of SEQ ID        NOs:40-46 or its complementary sequence.    -   (2) an influenza virus A probe including an oligo-nucleotide        selected from the non-structural protein gene region. For        example, the oligo-nucleotide can be one of SEQ ID NOs: 47-49 or        its complementary sequence.    -   (3) an influenza virus B probe including an oligo-nucleotide        selected from the hemagglutinin gene region. For example, the        oligo-nucleotide can be one of SEQ ID NOs:50-52 or its        complementary sequence.

The probe set of this invention can further contain one or more of thefollowing:

-   -   (1) a human parainfluenza virus 1 probe, e.g., a probe including        one of oligo-nucleotides SEQ ID NOs:28-33 or its complementary        sequence.    -   (2) a human parainfluenza virus 2 probe, e.g., a probe including        one of oligo-nucleotides SEQ ID NOs:34-36 or its complementary        sequence.    -   (3) a human parainfluenza virus 3 probe, e.g., a probe including        one of oligo-nucleotides SEQ ID NOs:37-39 or its complementary        sequence.    -   (4) an adenovirus probe, e.g., a probe including one of        oligo-nucleotides SEQ ID NOs: 53-57 or its complementary        sequences.

Also within the scope of this invention is a method of simultaneouslydetecting respiratory viruses using a PCR primer set, a set of amplifiednucleic acids, or a probe set described above. Among the viruses to bedetected are human parainfluenza virus 2 and adenovirus, and optionally,one or more of the following: human parainfluenza virus 1, humanparainfluenza virus 3, respiratory syncytial virus, influenza virus A,and influenza virus B. The method includes (1) providing a nucleic acidfrom a sample suspected of containing a virus to be detected, (2)amplifying the nucleic acid with one of the PCR primer sets describedabove, and (3) detecting amplification products. Detection of anamplification product specific for a target virus indicates the presenceof the target virus, and can be achieved by hybridizing theamplification products to one of the probe sets described above.

Further within the scope of this invention is a kit for simultaneousdetection of respiratory viruses including human parainfluenza virus 2and adenovirus, and optionally, one or more of the following: humanparainfluenza virus 1, human parainfluenza virus 3, respiratorysyncytial virus, influenza virus A, and influenza virus B. The kitcontains a PCR primer set, a set of amplified nucleic acids, a probe setdescribed above, or any combination thereof. It may include othercomponents such as a DNA polymerase, a PCR buffer, or a solid support onwhich one or more specific probes are immobilized.

The present invention enables one to simultaneously detect as many asseven common respiratory viruses. The details of one or more embodimentsof the invention are set forth in the accompanying description below.Other advantages, features, and objects of the invention will beapparent from the description, and from the claims.

DETAILED DESCRIPTION

The present invention relates to simultaneous detection of tworespiratory viruses, human parainfluenza virus 2 and adenovirus.Specifically, a nucleic acid template prepared from a sample suspectedof containing human parainfluenza virus 2 or adenovirus is amplifiedwith a set of PCR primers, which contains a pair of human parainfluenzavirus 2 primers and a pair of adenovirus primers. The amplificationproduct, if any, is detected by either gel electrophoresis and staining,or by probe hybridization. Detection of an amplification productspecific for human parainfluenza virus 2 or adenovirus indicates thepresence of that virus in the sample. Optionally, the set of PCR primerscan contain one or more additional primer pairs for detecting otherrespiratory viruses such as human parainfluenza virus 1, humanparainfluenza virus 3, respiratory syncytial virus, influenza virus A,and influenza virus B.

The nucleic acid template can be DNA (e.g., a genomic fragment or arestriction fragment) or RNA, in a purified or unpurified form. It canalso be obtained from a biological sample, e.g., a specimen from apatient having symptoms of respiratory infection.

The present invention features PCR primer pairs which can be used forsimultaneously detecting the seven respiratory viruses mentioned above.The primer pairs for each virus are selected by analyzing virussequences in GenBank using the DNAstar program (DNASTAR Inc., Madison,Wis. 53715, U.S.A.). A conserved region is first identified, followed byselection of primer pairs from this region. Each primer pair can betested in a PCR using a nucleic acid template prepared from a patientdiagnosed with infection of a virus to be detected to ensure that aspecific amplification product is produced. When a primer pair iscombined with other primer pairs for simultaneous detection of multipleviruses, the specificity of the primer pair should not be affected bythe presence of other primer pairs, i.e., the same amplification productis produced. Further, a primer pair selected for one virus preferablydoes not cause non-specific amplification of a nucleic acid templateprepared from another virus, and amplification products specific fordifferent viruses should be of different lengths.

Examples of PCR primer pairs which can be used for simultaneouslydetecting the seven respiratory viruses mentioned above are as follows:

-   -   (1) human parainfluenza virus 1 primer pairs selected from a        conserved hemagglutinin-neuraminidase gene region. For example,        forward primer PIV1-f834 and reverse primer PIV1-r1049, forward        primer PIV1-f849 and reverse primer PIV1-r1049, and forward        primer PIV1-f834 and reverse primer PIV1-r1099.    -   (2) human parainfluenza virus 2 primer pairs selected from a        conserved hemagglutinin-neuraminidase gene region. For example,        forward primer PIV2-f929 and reverse primer PIV2-r1182, and        forward primer PIV2-f1015 and reverse primer PIV2-r1182.    -   (3) human parainfluenza virus 3 primer pairs selected from a        conserved hemagglutinin-neuraminidase gene region. For example,        forward primer PIV3-f774 and reverse primer PIV3-r960, forward        primer PIV3-f774 and reverse primer PIV3-r1059, and forward        primer PIV3-f904 and reverse primer PIV3-r1059.    -   (4) respiratory syncytial virus primer pairs selected from a        conserved non-structural protein 2 gene region. For example,        forward primer RSV-f417 and reverse primer RSV-r641, and forward        primer RSV-f1351 and reverse primer RSV-r1540.    -   (5) influenza virus A primer pairs selected from a conserved        non-structural protein gene region. For example, forward primer        INFA-f1 and reverse primer INFA-r1, forward primer INFA-f2 and        reverse primer INFA-r2, and forward primer INFA-f1 and reverse        primer INFA-r2.    -   (6) influenza virus B primer pairs selected from a conserved        hemagglutinin protein gene region. For example, forward primer        INFB-92f and reverse primer INFB-384r, and forward primer        INFB-540f and reverse primer INFB-820r.    -   (7) adenovirus primer pairs selected from a conserved Hexon        protein gene region. For example, forward primer ADV-f1 and        reverse primer ADV-r2, and forward primer ADV-f2 and reverse        primer ADV-r1.

The sequence of each primer is listed in Table 1 below in Example 1.Typically, a primer is 14-40 nucleotides in length (PCR ApplicationManual, Boehringer Mannheim, 1995, page 37). In this invention, specificvirus sequences can be added to either the 5′-end or the 3′-end of eachprimer; non-specific sequences can be added to the 5′-end of eachprimer. An example of a non-specific sequence is a sequence containing arestriction site. Addition of such a sequence facilitates cloning of theamplification product.

The present invention also features probes chosen from the regionsamplified with primer pairs described above using the DNAstar program.Examples of the probes are as follows:

-   -   (1) human parainfluenza virus 1 probes P1-1, P1-2, P1-3,        PIV1-P4, PIV1-P5, and PIV1-P6;    -   (2) human parainfluenza virus 2 probes P2-1, P2-2, and P2-3;    -   (3) human parainfluenza virus 3 probes P3-1, P3-2, and P3-3;    -   (4) respiratory syncytial virus probes R-1, R-2, R-3, R-10, R-4,        R-71, and R-72;    -   (5) influenza virus A probes A1, A2, and A3;    -   (6) influenza virus B probes B1, B2, and B3;    -   (7) adenovirus probes D-1, D-2, D-3, ADV-P4, and ADV-P5.

The sequence of each probe is listed in Table 3 below in Example 3.These probes, and longer probes containing them and having 20-1000(e.g., 20-500, 20-200, and 20-50) nucleotides in length can be used fordetecting the seven viruses mentioned above by hybridizing tounamplified target virus nucleic acids or target virus nucleic acidsamplified with the above-described primer pairs. For instance, theamplification products described above are examples of such longerprobes. GenBank search indicates that the nucleic acid sequencesamplified with the primer pairs described above are specific for each ofthe seven viruses. When a probe is combined with other probes forsimultaneous detection of multiple viruses, the specificity of the probeshould not be affected by the presence of other probes, i.e., it stillhybridizes to the target virus nucleic acid. Preferably, a probeselected for one virus does not hybridize to a nucleic acid preparedfrom another virus.

The probes can be immobilized on the surface of a solid support, such asa membrane (a nylon-membrane or a nitrocellulose membrane), a glass, ora plastic polymer. Immobilization of probes to a membrane can beachieved by baking at 80° C. or UV cross-linking. The probes can also becovalently linked to a material (e.g., poly-lysine) coated on thesurface of a glass. In addition, a novel method of immobilizing probeson a plastic polymer has recently been developed. See U.S. applicationSer. No. 09/906,207. Alternatively, the probes can be synthesized denovo at precise positions on a solid substrate. See Schena et al., 1995,Science 270: 467; Kozal et al., 1996, Nature Medicine 2(7): 753; Chenget al., 1996, Nucleic Acids Res. 24(2): 380; Lipshutz et al., 1995,BioTechniques 19(3): 442; Pease et al., 1994, Proc. Natl. Acad. Sci. USA91: 5022; Fodor et al., 1993, Nature 364: 555; and Fodor et al., WO92/10092.

A target amplification product described above can be detected bybinding it to an immobilized probe. To facilitate detection, a labeledamplification product can be generated with a labeled amplificationprimer. Alternatively, the labeling can be done, chemically orenzymatically, after amplification. Examples of labeling reagentsinclude, but are not limited to, a fluorescent molecule (e.g.,fluorescein and rhodamine), a radioactive isotope (e.g., ³²P and ¹²⁵I),a calorimetric reagent, and a chemiluminescent reagent. Biotin anddigoxgenin are frequently used for colorimetric detection on a membraneor a plastic polymer. Fluorescent labels, such as Cy3 and Cy5, arewidely used for detection on a glass. In addition, artificial taggingtails (e.g., a protein or its antibody) can be conjugated to the 5′-endof the primers or either end of the probes. See Stetsenko and Gait,2000, J. Org. Chem. 65(16): 4900.

The specificity of the virus detection method of this invention isunexpectedly high. When primer pairs specific for different viruses aremixed together for simultaneous detection of the viruses, each virusnucleic acid template is only amplified with a primer pair selected forthat particular virus. There is no non-specific amplification caused bythe presence of other primer pairs. Likewise, when probes specific fordifferent viruses are mixed together for simultaneous detection of theviruses, each target virus nucleic acid only hybridizes to a probeselected for that particular virus. There is no non-specifichybridization between a target virus nucleic acid and probes selectedfor other viruses.

Also within the scope of this invention is use of the above-describedsequences specific for the seven viruses in combination with otherspecies-specific nucleic acid sequences for simultaneous identificationof even more microorganisms.

Furthermore, at positions where single nucleotide polymorphisms occur,nucleotide variations are allowed in primers and probes described inthis invention. As single nucleotide polymorphisms may be associatedwith a particular genotype or phenotype, these primers and probes can beused to distinguish and categorize different virus strains.

The specific examples below are to be construed as merely illustrative,and not limitative of the remainder of the disclosure in any waywhatsoever. Without further elaboration, it is believed that one skilledin the art can, based on the description herein, utilize the presentinvention to its fullest extent. All publications recited herein arehereby incorporated by reference in their entirety.

EXAMPLE 1 Amplification and Detection of a Specific Respiratory Virus

A. Design of Primers

(1) Human Parainfluenza Virus 1 (HPIV 1) Primers

HPIV 1 sequences from GenBank Accession Nos u01075, af016281, m31228,m86780, m86781, m86783, m86785, m86787, m86791, m91648, u01073, u01074,af016280, u70948u01079, u01081, u01083, u01085, u70936, u10938, u70940,u70942, u70944, u70946, and u01077 were analyzed using the DNAstarprogram. A conserved region was identified in thehemagglutinin-neuraminidase (HN) gene area. Two forward primers(PIV1-f834 and PIV1-f849) and two reverse primers (PIV1-r1049 andPIV1-r1099) were selected from this conserved region (Table 1).

(2) Human Parainfluenza Virus 2 (HPIV 2) Primers

HPIV 2 sequences from GenBank Accession Nos af039930, af039931,af039932, af039934, af039937, x57559, af213353, af213354, d00865, andaf213352 were analyzed using the DNAstar program. A conserved region wasidentified in the HN gene area. Two forward primers (PIV2-f929 andPIV2-f1015) and one reverse primer (PIV2-r1182) were selected from thisconserved region (Table 1).

(3) Human Parainfluenza Virus 3 (HPIV 3) Primers

HPIV 3 sequences from GenBank Accession Nos Z26532, L25350, M17641,M18759, M18760, M18761, M18762, M18763, M18764, M20402,M21649,NC_(—)001796, U51116, Z11575, aB012132, af039922, AF039924,AF039925, AF039926, AF039927, AF039929, AF039933, and AF039936 wereanalyzed using the DNAstar program. A conserved region was identified inthe HN gene area. Two forward primers (PIV3-f774 and PIV3-f904) and tworeverse primers (PIV3-r960 and PIV3-r1059) were selected from thisconserved region (Table 1).

(4) Respiratory Syncytial Virus (RSV) Primers

RSV sequences from GenBank Accession Nos af035006, 63644, 50362, 50363,11486, 74568, C_(—)001803, 39661, 39662, 00001, f013255, C_(—)001781,F013254, and 00736 were analyzed using the DNAstar program. A conservedregion was identified in the non-structural protein 2 (NS 2) gene area.Two forward primers (RSV-f417 and RSV-fl 351) and two reverse primers(RSV-r641 and RSV-r1540) were selected from this conserved region (Table1).

(5) Influenza Virus A (INF A) Primers

INF A sequences from GenBank Accession Nos m12594, k00576, m12592,m12590, d30673, j02150, x52146, u08862, u65674, and u65670 were analyzedusing the DNAstar program. A conserved region was identified in thenon-structural protein (NS) gene area. Two forward primers (INFA-f1 andINFA-f2) and two reverse primers (INFA-r1 and INFA-r2) were selectedfrom this conserved region (Table 1).

(6) Influenza Virus B (INF B) Primers

INF B sequences from GenBank Accession Nos x13552, x00897, m18384,u70384, x13550, af101071, m58422, m58421, m65170, and k02713 wereanalyzed using the DNAstar program. A conserved region was identified inthe hemagglutinin protein (HA) gene area. Two forward primers (INFB-92fand INFB-540f) and two reverse primers (INFB-384r and INFB-820r) wereselected from this conserved region (Table 1).

(7) Adenovirus (ADV) Primers

ADV sequences from GenBank Accession Nos x67709, ab053166, x76549,x84646, af065066,j01917, and j01966 were analyzed using the DNAstarprogram. A conserved region was identified in the Hexon protein (Hex)gene area. Two forward primers (ADV-f1 and ADV-f2) and two reverseprimers (ADV-r1 and ADV-r2) were selected from this conserved region(Table 1).

TABLE 1 Primer sequences PIV1-f834 5′-CTGTAATAGCTGCAGGAACAAG-3′ (SEQ IDNO:1) PIV1-f849 5′-ACAAGGGGTTATCAGTTATGCTC-3′ (SEQ ID NO:2) PIV1-r10495′-TTCAATYTTTATCCCRCTTCCTAC-3′ (SEQ ID NO:3) PIV1-r10995′-CCTTGGAGCGGAGTTGTTA-3′ (SEQ ID NO:4) PIV2-f9295′-GCTGTTCAGTCACTGCTATACC-3′ (SEQ ID NO:5) PIV2-f10155′-GATCTAGCTGAACTGAGACTTGC-3′ (SEQ ID NO:6) PIV2-r11825′-TATGAGACCACCATATACAGGAAA-3′ (SEQ ID NO:7) PIV3-f7745′-CTGTAAACTCAGACTTGGTACCTG-3′ (SEQ ID NO:8) PIV3-f9045′-AGTTGATGAAAGATCAGATTATGC-3′ (SEQ ID NO:9) PIV3-r9605′-ATATCAAGTACAATATCTTCTATGCC-3′ (SEQ ID NO:10) PIV3-r10595′-CCTGGTCCAACAGATGGGTAT-3′ (SEQ ID NO:11) RSV-f4175′-GYATTGGCATTAAGCCTACAA-3′ (SEQ ID NO:12) RSV-f13515′-GGATTGTTTATGAATGCCTATGGT-3′ (SEQ ID NO:13) RSV-r6415′-AACTTGACTTTGCTAAGAGCCAT-3′ (SEQ ID NO:14) RSV-r15405′-TTGGRTTGTTCAATATATGGTAGA-3′ (SEQ ID NO:15) INFA-f15′-CACTTAAAATGACCATGGCCTC-3′ (SEQ ID NO:16) INFA-f25′-CGAAATTTCACCATTGCCTTC-3′ (SEQ ID NO:17) INFA-r15′-GAAGGCTTAGGTGAAATTTCGC-3′ (SEQ ID NO:18) INFA-r25′-GTCTCACTTCTTCAATCAGCCA-3′ (SEQ ID NO:19) INFB-92f5′-CTGGGATAACATCKTCAAACTC-3′ (SEQ ID NO:20) INFB-540f5′-AACAATGGCTTGGGCTG-3′ (SEQ ID NO:21) INFB-384r5′-TGTTCTGTCGTGCATTATAGG-3′ (SEQ ID NO:22) INFB-820r5′-CAACAATTCTRCCGCTTT-3′ (SEQ ID NO:23) ADV-f1 5′-CCACCTTCTTCCCCAT-3′(SEQ ID NO:24) ADV-f2 5′-AACATGACCAARGACTGGT-3′ (SEQ ID NO:25) ADV-r15′-CTCATKGGCTGGAAGTT-3′ (SEQ ID NO:26) ADV-r2 5′-GAACCAGTCYTTGGTCATGT-3′(SEQ ID NO:27)B. Preparation of a Virus Culture

Clinical samples were kindly provided by Dr. Shin-Ru-Shih (Chang GungUniversity, Tao Yuan, Taiwan): HPIV 1 (sample numbers 580 and 4056),HPIV 2 (sample numbers 4855 and 5088), HPIV 3 (sample numbers 3116 and3229), RSV (sample numbers 3116 and 3229), INF A (sample numbers NSW,PC, and DR), INF B (sample numbers 95, 96, 97, 98, 01), and ADV (samplenumbers 5456, 608, and 819).

Throat swab specimens were collected from patients showing symptoms ofvirus infection, and were transferred to Hank's solution containing 0.2%BPA (p-borono-L-phenylalanine), 100 units of penicillin, 100 μgstreptomycin, and 1.25 μg fungizone. Samples were kept at roomtemperature for 0.5-3 hours in emergency room before refrigerated at 4°C. or −80° C.

Monkey Kidney-2 cells were cultivated in a cell culture mediumcontaining 2% FBS-MEM, 100 units of penicillin, 100 μg streptomycin, and1.25 μg fungizone. Clinical specimens, 100 μl each, were inoculated intothe cell culture medium and were incubated at 37□, with CO₂, for 7-10days.

C. Nucleic Acid Extraction and RT-PCR

Virus culture was centrifuged. Nucleic acids were isolated from 50 μl ofthe supernatant using the High Pure Viral Nucleic Acid Purification Kit(Roche), and were suspended in 20 μl DEPC-treated H₂O.

Two microliters of the nucleic acid solution were mixed with 1 μl ofrandom primers Pd(N)₆ (Roche, 5 μg/μl). The mixture was incubated at 72□for 10 min, and was stored at 4 □.

The Ready-To-Go RT-PCR Beads (Amersham Pharmacia Biotech Inc., U.S.A.)was dissolved in 35 μl DEPC-treated H₂O. 7 μl of the solution were addedto the pretreated nucleic acid mixture described above. The finalmixture was incubated at 42□ for 45 min. Note that ADV is a DNA virus,thus the reverse transcription is not applicable.

Each PCR tube contained 1 μl of 10X Taq DNA polymerase buffer, 0.3 μl of25 mM MgCl₂, 0.8 μl of 2.5 mM dNTPs (Promega, Madison, Wis., U.S.A.),0.2 μl of 100 μM forward primer, 0.2 μl of 100 M reverse primer, 0.2 μlformamide, 5 μl of reverse transcription mixture, and 0.1 μl Taq DNApolymerase (5 units/μl). dH₂O was added to the mixture to bring thefinal volume to 10 μl.

Amplification was carried out using Peltier-effect Thermal Cyclers(PTC-100, MJ Research Inc., MA, U.S.A.) as follows: 95□ for 5 min; 35cycles of 95□ for 40 sec, 50□ for 40 sec, and 72□ for 40 sec; and afinal extension at 72□ for 5 min.

D. Detection of Amplification Products

Five microliters of amplified products were analyzed by electrophoresison a 2% agarose gel in TAE buffer (Tris-HCl, pH 8.0, 1 mM EDTA).Amplification products were detected by staining the agarose gel withethidium bromide.

Unexpectedly, specific amplification products were detected as follows:

-   -   (1) HPIV 1: a218 bp fragment from amplification with primer set        f834 and r1049, a 201 bp fragment from amplification with primer        set f849 and r1049, and a 268 bp fragment from amplification        with primer set f834 and r1099. Amplification with primer set        f849 and r1099 produced a 251 bp non-specific fragment.    -   (2) HPIV 2: a 254 bp fragment from amplification with primer set        f929 and r1182, and a 168 bp fragment from amplification with        primer set f1015 and r1182.    -   (3) HPIV 3: a 187 bp fragment from amplification with primer set        f774 and r960, a 286 bp fragment from amplification with primer        set f774 and r1059, and a 156 bp fragment from amplification        with primer set f904 and r1059. Amplification with primer set        f904 and r960 failed to produce a 57 bp fragment.    -   (4) RSV: a 222 bp fragment from amplification with primer set        f417 and r641, and a 194 bp fragment from amplification with        primer set f1351 and r1540.    -   (5) INF A: a 268 bp fragment from amplification with primer set        INFA-f1 and INFA-r1, a 252 bp fragment from amplification with        primer set INFA-f2 and INFA-r2, and a 499 bp fragment from        amplification with primer set INFA-f1 and INFA-r2.    -   (6) INF B: a 293 bp fragment from amplification with primer set        92f and 384r, and a 275 bp fragment from amplification with        primer set 540f and 820r. However, the 275 bp band is not as        strong as the 293 bp band.    -   (7) ADV: a 556 bp fragment from amplification with primer set        ADV-f1 and ADV-r1, a 449 bp fragment from amplification with        primer set ADV-f1 and ADV-r2, and a 128 bp fragment from        amplification with primer set ADV-f2 and ADV-r1.

EXAMPLE 2 Simultaneous Detection of Seven Respiratory Viruses on anAgarose Gel

Clinical samples used in this example were PIV 1 number 580, PIV2 number5129, PIV3 number 1057, RSV number 3167, INF A number DR, INF B number01, and ADV number 5456, provided by Dr. Shin-Ru-Shih (Chang GungUniversity, Tao Yuan, Taiwan).

All steps were carried out as described in Example 1, except that anoligo-nucleotide primer mixture was used for amplification instead of asingle pair of primers. The oligo-nucleotide primer mixture containedseven pairs of primers, one for each of the seven viruses (Table 2). Thefinal concentration of each primer was 1 μM.

TABLE 2 Primers used for muliplex PCR PIV1-f8345′-CTGTAATAGCTGCAGGAACAAG-3′ (SEQ ID NO:1) PIV1-r10995′-CCTTGGAGCGGAGTTGTTA-3′ (SEQ ID NO:4) PIV2-f10155′-GATCTAGCTGAACTGAGACTTGC-3′ (SEQ ID NO:6) PIV2-r11825′-TATGAGACCACCATATACAGGAAA-3′ (SEQ ID NO:7) PIV3-f9045′-AGTTGATGAAAGATCAGATTATGC-3′ (SEQ ID NO:9) PIV3-r10595′-CCTGGTCCAACAGATGGGTAT-3′ (SEQ ID NO:11) RSV-f4175′-GYATTGGCATTAAGCCTACAA-3′ (SEQ ID NO:12) RSV-r6415′-AACTTGACTTTGCTAAGAGCCAT-3′ (SEQ ID NO:14) INFA-f25′-CGAAATTTCACCATTGCCTTC-3′ (SEQ ID NO:17) INFA-r25′-GTCTCACTTCTTCAATCAGCCA-3′ (SEQ ID NO:19) INFB-92f5′-CTGGGATAACATCKTCAAACTC-3′ (SEQ ID NO:20) INFB-384r5′-TGTTCTGTCGTGCATTATAGG-3′ (SEQ ID NO:22) ADV-f1 5′-CCACCTTCTTCCCCAT-3′(SEQ ID NO:24) ADV-r1 5′-CTCATKGGCTGGAAGTT-3′ (SEQ ID NO:26)

Unexpectedly, a specific amplification product was detected in eachsample, i.e., (1) a 268 bp fragment from amplification of the HPIV 1template with primer set f834 and r1099, (2) a 168 bp fragment fromamplification of the HPIV 2 template with primer set f1015 and r1182f,(3) a 156 bp fragment from amplification of the HPIV 2 template withprimer set f904 and r1059, (4) a 222 bp fragment from amplification ofthe RSV template with primer set f417 and r641, (5) a 252 bp fragmentfrom amplification of the INF A template with primer set INFA-12 andINFA-r2, (6) a 293 bp fragment from amplification of the INF B templatewith primer set 92f and 84r, and (7) a 556 bp fragment fromamplification of the ADV template with primer set ADV-f1 and ADV-r1.

EXAMPLE 3 Simultaneous Detection of Seven Respiratory Viruses on anArray Chip

Sample preparation and RT-PCR were carried out as described in Example2, except that all primers were labeled with biotin at the 5′-end.Amplification products were detected on an array chip described below.

A. Design of Probes

Probes were selected from the amplified region of each virus genomeusing the DNAstar program (Table 3).

TABLE 3 Probe sequences HPIV 1 probes P1-15′-CTCCYTGCCYACTGTRAATGAGACTA-3′ (SEQ ID NO:28) P1-25′-CGAGTGAAGGTATAGAAGAYTTAGTATTTGACA-3′ (SEQ ID NO:29) P1-35′-CTCAAGGGAAAGACCAAATCTCATCG-3′ (SEQ ID NO:30) PIV1-P45′-GCTGCAGGAACAAGGGGTTATCAGTTATGC-3′ (SEQ ID NO:31) PIV1-P55′-GTGTAGGAAGRGGGATAAAYATTGAA-3′ (SEQ ID NO:32) PIV1-P65′-CCTTGGAGCGGAGTTGTTAAGCCACCG-3′ (SEQ ID NO:33) HPIV 2 probes P2-15′-GATCTAGCTGAACTGAGACTTGCTTTCTATTATTAT-3′ (SEQ ID NO:34) P2-25′-TCATATCTCTTCCAAAYACAACAGGGCA-3′ (SEQ ID NO:35) P2-35′-TGCAGTYGGAAGCGGGATCTATC-3′ (SEQ ID NO:36) HPIV 3 probes P3-15′-TGCATCATCAGGCATAGAAGATATTGTAC-3′ (SEQ ID NO:37) P3-25′-ATYATGATGGYTCAATCTCAACAACAAG-3′ (SEQ ID NO:38) P3-35′-ATCTCAACAACAAGATTTAAGAAYAATAAYATAA-3′ (SEQ ID NO:39) RSV probes R-1(RSV A) 5′-TAAGCCTACAAAGCAYACTCCCATAATA-3′ (SEQ ID NO:40) R-2 (RSV B)5′-AAATATGACCTCAACCCGTAAATTCCAA-3′ (SEQ ID NO:41) R-3 (RSV B)5′-AACCCAWCCAAACYAAGCTATTCC-3′ (SEQ ID NO:42) R-10 (RSV A)5′-GATGGAGCCTGAAAATTATAGTAATTTAAAATTAAGGAG-3′ (SEQ ID NO:43) R-4 (RSV B)5′-CAAACAACARTGCTCAAYAGTTAAGAAGGA-3′ (SEQ ID NO:44) R-71 (RSV A)5′-AGGAGAGATATAAGATGAAAGATGGGGC-3′ (SEQ ID NO:45) R-72 (RSV A)5′-AGGAGAGACATAAGATAGAAGATGGGGC-3′ (SEQ ID NO:46) INF A probes A15′-CGGAGGACTTGAATGGARTGATAACAC-3′ (SEQ ID NO:47) A25′-TCTACAGAGATTCGCTTGGRGAAGCAG-3′ (SEQ ID NO:48) A35′-GACMTCCACTYACTCCAAAACAGAAAC-3′ (SEQ ID NO:49) INF B probes B15′-TGTGATACCACTGACAACAACACCWAC-3′ (SEQ ID NO:50) B25′-GCAAATCTCAAAGGAACAAARACYAG-3′ (SEQ ID NO:51) B35′-AAACTATGCCCAAMSTGTYTCAACTGY-3′ (SEQ ID NO:52) ADV probes D-15′-TCCCCATGGCBCACAACACNGC-3′ (SEQ ID NO:53) D-25′-TCKCGCAACTGGGCRGCYTT-3′ (SEQ ID NO:54) D-35′-AACCACACYTTYAAGAAGGTSKCCATC-3′ (SEQ ID NO:55) ADV-P45′-AACATGACCAARGACTGGTTCCTGGT-3′ (SEQ ID NO:56) ADV-P55′-TACAAYATYGGMTACCAGGGCTTYTA-3′ (SEQ ID NO:57)B. Spotting and Hybridization

Probes were spotted on a DR. Polymer™ Chip. Additional 5 to 20-mers ofpoly-T were attached to each of the probes before spotting. The probeswere UV cross-linked to the surface of a DR. Polymer™ Chip. 4 positivedetection controls, 4 positive PCR controls, 4 positive hybridizationcontrols, and 4 negative controls were also spotted on the polymer chip.

Prior to hybridization, 8 μl of PCR products were denatured for 5minutes and mixed with 392 μl of DR. Hyb™ Hybridization Buffer-E. Themixture was added to the DR. Polymer™ Chip in a hybridization chamberand incubated at 50° C. for 1 hour. The chip was subsequently washedwith 500 μl DR. Wash Buffer 5 times.

C. Detection

Four hundred microliters of DR. Block Buffer were mixed with 0.2 μlstreptavidin-AP (alkaline phosphatase). The mixture was added to the DR.Polymer™ Chip in a hybridization chamber, and was incubated for 30minutes at room temperature. The chip was subsequently washed with DR.Wash Buffer 5 times.

Three hundred and ninety-two microliters of DR. Detection Buffer weremixed with 8 μl NBT/BCIP. The mixture was added to the DR. Polymer™ Chipin a hybridization chamber, and was incubated for 10 minutes in a darkroom. The chip was subsequently washed twice with DR. Wash Buffer priorto color development.

Unexpectedly, each probe specifically designed for a particular viruswas able to capture the amplification product produced from the templateisolated from the target virus sample, i.e., (1) color developed atP1-1, P1-2, P1-3, PIV1-P4, PIV1-P5, and PIV1-P6 spots when the probeswere hybridized to HPIV 1 amplification products; (2) color developed atP2-1P2-2, and P2-3 spots when the probes were hybridized to HPIV 2amplification products; (3) color developed at P3-1, P3-2, and P3-3spots when the probes were hybridized to HPIV 3 amplification products;(4) color developed at R-1, R-2, R-3, R-10, R-4, and a mixture of R-71and R-72 spots when the probes were hybridized to RSV amplificationproducts; (5) color developed at A1, A2, and A3 spots when the probeswere hybridized to INF A amplification products; (6) color developed atB1, B2, and B3 spots when the probes were hybridized to INF Bamplification products; and (7) color developed at D-1, D-2, D-3,ADV-P4, and ADV-P5 spots when the probes were hybridized to ADVamplification products.

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the scope of thefollowing claims.

1. A set of nucleic acids comprising: a first pair of primers, bothcontaining oligo-nucleotides selected from thehemagglutinin-neuraminidase gene region of human parainfluenza virus 2,and the oligo-nucleotides in the first pair of primers being,respectively, SEQ ID NOs:5 and 7, or SEQ ID NOs:6 and 7; a second pairof primers, both containing oligo-nucleotides selected from the hexongene region of adenovirus, the oligo-nucleotides in the second pair ofprimers being, respectively, SEQ ID NQs:24 and 26, SEQ ID NOs:24 and 27,or SEQ ID NOs:25 and 27; and a third pair of primers, both containingoligo-nucleotides selected from the non-structural protein 2 gene regionof respiratory syncytial virus, the oligo-nucleotides in the third pairof primers being, respectively, SEQ ID NOs:12 and 14, or SEQ ID NOs:13and 15, wherein each nucleic acid is 14-40 nucleotides in length, andeach primer is a single compound.
 2. The set of nucleic acids of claim1, further comprising: a fourth pair of primers containing,respectively, oligo-nucleotides SEQ ID NOs:1 and 3, SEQ ID NOs:2 and 3,or SEQ ID NOs:1 and 4; a fifth pair of primers containing, respectively,oligo-nucleotides SEQ ID NOs:8 and 10, SEQ ID NOs:8 and 11, or SEQ IDNOs:9 and 11; a sixth pair of primers containing, respectively,oligo-nucleotides SEQ ID NOs:16 and 18, or SEQ ID NOs:17 and 19; or aseventh pair of primers containing, respectively, oligo-nucleotides SEQID NO:20 and 22, or SEQ ID NOs:21 and 23, or a combination thereof.
 3. Aset of nucleic acids comprising: a first nucleic acid containing a firstoligo-nucleotide selected from the non-structural protein 2 gene regionof respiratory syncytial virus; a second nucleic acid containing asecond oligo-nucleotide selected from the hemagglutinin gene region ofinfluenza virus B; and a third nucleic acid containing a thirdoligo-nucleotide selected from the non-structural protein gene region ofinfluenza virus A, wherein each oligo-nucleotide is a single compoundselected from the group consisting of SEQ ID NOs:40-52 and sequencescomplementary thereto, and each nucleic acid is 20-200 nucleotides inlength.
 4. The set of nucleic acids of claim 3, wherein each nucleicacid has 20-50 nucleotides in length.
 5. The set of nucleic acids ofclaim 3, further comprising a nucleic acid containing anoligo-nucleotide selected from the group consisting of SEQ ID NQs:28-39,53-57, and sequences complementary thereto, wherein each nucleic acidhas 20-200 nucleotides in length.
 6. The set of nucleic acids of claim5, wherein each nucleic acid has 20-50 nucleotides in length.